Cleavable conjugates of antibiotics and an antibacterial cell-penetrating peptide

ABSTRACT

The present disclosure relates to novel cleavable conjugates of antibiotics and an antibacterial cell-penetrating peptide, and methods to make and use the novel cleavable conjugates of antibiotics and an antibacterial cell-penetrating peptide.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 16/050,472 filed Jul. 31, 2018, which is related toand claims the benefits of U.S. Provisional Application Ser. No.62/539,570, filed Aug. 1, 2017, the contents of which are incorporatedherein entirely.

GOVERNMENT RIGHTS

This invention was made with government support under CHE1012316 awardedby the National Science Foundation. The United States government hascertain rights in the invention.

STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTORS OR JOINTINVENTORS UNDER 37 C.F.R. 1.77(B)(6)

Jean A. Chmielewski and Mohamed Seleem, the inventors or joint inventorsof the present disclosure, publicly disclosed information related to thepresent disclosure in article Anna Brezden, etc., Dual targeting ofintracellular pathogenic bacteria with a cleavable conjugate ofkanamycin and an antibacterial cell-penetrating peptide, J. Am. Chem.Soc., 2016, 138, 10945-10949. The article was first published online onAug. 5, 2016, which is less than one year from the filing date of theU.S. Provisional Application Ser. No. 62/539,570, filed Aug. 1, 2017.The other five listed co-authors Anna Brezden, Mohamed F. Mohamed,Manish Nepal, John S. Harwood, and Jerrin Kuriakose of the article arenot inventors for the present disclosure because the five listedco-authors only provided supervised contributions instead of providinginventive contribution. A copy of a print out of the article is providedon a concurrently filed Information Disclosure Statement (IDS).

TECHNICAL FIELD

The present disclosure relates to novel cleavable conjugates ofantibiotics and an antibacterial cell-penetrating peptide, and methodsto make and use the novel cleavable conjugates of antibiotics and anantibacterial cell-penetrating peptide.

BACKGROUND

This section introduces aspects that may help facilitate a betterunderstanding of the disclosure. Accordingly, these statements are to beread in this light and are not to be understood as admissions about whatis or is not prior art.

The emergence of antibiotic resistance is a notorious problem worldwide.In the United States alone, antibiotic-resistant bacteria infect atleast two million people, killing 23,000 patients annually. Currently,members of the ESKAPE pathogens (Enterococcus faecium, Staphylococcusaureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonasaeruginosa, and Enterobacter species) represent one of the mostsignificant threats to human health as strains exhibit resistance tomultiple antibiotic classes. Compounding the situation further, globalreports of extensively drug-resistant (XDR) and pandrug-resistant (PDR)isolates of P. aeruginosa and A. baumannii have been increasing. Theability of these resistant pathogens to form biofilms that are highlytolerant to antibiotics further aggravates the situation, ultimatelyleading to persistent and recurring infections.

Aminoglycosides are a class of valuable antibiotics that are used in thetreatment of several microbial infections. Aminoglycosides bind to the16S rRNA component of the bacterial ribosome, leading to mistranslationand bacterial death. However, the worldwide epidemic of resistance toaminoglycosides, to kanamycin in particular, has diminished theirwidespread clinical use.

Hence, the development of therapeutic alternatives that have novelmechanisms of action are urgently needed in order to effectively addressthis problem.

SUMMARY

The present invention provides novel cleavable conjugates of antibioticsand an antibacterial cell-penetrating peptide that may provide novelmechanisms of action to better address the antibiotic resistance issue.Specifically, the novel cleavable conjugates of antibiotics and anantibacterial cell-penetrating peptide of the present disclosure arerepresented in the formula below:

or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt,derivative or prodrug thereof, wherein

-   -   R¹ is an antibiotic moiety;    -   R² and R³ are each independently H, a C₁-C₈ branched or        unbranched alkyl chain, or a C₃-C₈ cyclic alkyl;    -   R⁴ is H, a C₁-C₈ branched or unbranched alkyl chain, or a C₃-C₈        cyclic alkyl;    -   L₃ is C₁-C₈ branched or unbranched alkyl chain, or a C₃-C₈        cyclic alkyl;    -   Z is a linker comprising a disulfide bond (S—S bond); and    -   n is 3-8.

In one embodiment, the present disclosure provides methods for treatinga patient, either mammal or animal, having a microbial infection withthe presently disclosed novel cleavable conjugates of antibiotics and anantibacterial cell-penetrating peptide, or a stereoisomer, tautomer,solvate, pharmaceutically acceptable salt, derivative or prodrugthereof.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to the embodimentsillustrated in the drawings, and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of this disclosure is thereby intended.

In the present disclosure the term “about” can allow for a degree ofvariability in a value or range, for example, within 10%, within 5%, orwithin 1% of a stated value or of a stated limit of a range.

As used herein, the term “salts” and/or “pharmaceutically acceptablesalts” refer to derivatives of the disclosed compounds wherein theparent compound is modified by making acid or base salts thereof.Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic groups such asamines; and alkali or organic salts of acidic groups such as carboxylicacids. Pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include those derived frominorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,phosphoric, and nitric; and the salts prepared from organic acids suchas acetic, propionic, succinic, glycolic, stearic, lactic, malic,tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, andisethionic, and the like.

As used herein, the term “nitrogen-protecting group” in the presentdisclosure may be any functional group that can make the amine nitrogento be protected as any form of carbamate, benzyl amine, amide,thioamide, sulfonamide, urea, or thiourea. The nitrogen-protecting groupmay include but is not limited to benzoyl, benzyloxycarbonyl,t-butoxycarbonyl, benzene sulfonyl, toluene sulfonyl, benzyl,benzhydryl, trityl, acetyl, or trifluoroacetyl.

As used herein, the term “antibiotics” refers to any anti-bacterialantibiotics, which are antibiotics that do not have activity againstviruses, fungi and other non-bacterial microbes. The anti-bacterialantibiotics include bactericidal antibiotics, which destroy bacteria,and bacteriostatic antibiotics which prevent bacteria from multiplying.The anti-bacterial antibiotics further include “narrow-spectrum”antibiotics which target particular types of bacteria, such as Gramnegative or Gram-positive bacteria, and broad spectrum antibiotics whichaffect a wide range of bacteria. Likewise, the anti-bacterialantibiotics include antibiotics for ingestion as well as antibiotics forintravenous administration which are often used to treat seriousinfections such as deep-seeded systemic infections, and antibiotics fortopical administration. The anti-bacterial antibiotics compriseantibiotics within the following presently recognized classes:aminoglycoside antibiotics, Ansamycins, Beta-lactam antibiotics(including the carbacephem, carbapenems, cephalosporins (first, second,third and fourth generations), monobactams and penicillins,Glycopeptides, Macrolides, lincosamides, Polypeptides, Quinolones,Sulphonamides, Tetracyclines, Cyclic lipopeptides, Glycylcyclines,Oxazolidinones, diaminopyrimidines, Nitrofurans, Rifamycins, antibioticpeptides, amphenicols, nitroimidazoles, streptogramins andphosphomycins.

As used herein, the particular term “aminoglycoside antibiotics” refersto a medicinal and bacteriologic category of traditional antibacterialtherapeutic agents that inhibit protein synthesis and contain as aportion of the molecule an amino-modified glycoside (sugar). The termcan also refer more generally to any organic molecule that containsamino sugar substructures. Aminoglycoside antibiotics may include but isnot limited to Gentamicin, Streptomycin, Kanamycin, Fradiomyctn,Paromomycin, Tobramycin, Netilmicin, Amikacin, Neomycin, Ribostamycin,Dibekacin, Sisomicin, Isepamicin, Bekanamycin, Astromicin, Plazomicin,or any derivative thereof.

As used herein, the term “antibiotic moiety” as referred in a chemicalstructure refers to the structure of an antibiotic agent that ischemically linked to the structure of another chemical or partialstructure of another chemical, through a chemical functional groups suchas an ester group, an amide group or an acyl amide group through thehydroxyl, amino or amide group of the antibiotic agent.

The term “cleavable” means that the compound of the present disclosurecan be cleaved from a disulfide bond (—S—S—).

The present invention provides novel cleavable conjugated cellpenetrating peptides. Accordingly, the present invention provides acompound of Formula I:

or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt,derivative or prodrug thereof, wherein

-   -   R¹ is an antibiotic moiety;    -   R² and R³ are each independently H, a C₁-C₈ branched or        unbranched alkyl chain, or a C₃-C₈ cyclic alkyl;    -   R⁴ is H, a C₁-C₈ branched or unbranched alkyl chain, or a C₃-C₈        cyclic alkyl;    -   L₁, L₂, L₃ are each independently C₁-C₈ branched or unbranched        alkyl chain, or a C₃-C₈ cyclic alkyl;    -   X is O or NR⁵, wherein R⁵ is H, C₁-C₈ branched or unbranched        alkyl chain, or a C₃-C₈ cyclic alkyl, or X combined with R¹        together is an antibiotic moiety; and    -   n is 3-8.

In one embodiment, the present invention provides a compound of FormulaI, wherein R¹ or R¹—X represents the moiety of an aminoglycosideantibiotics or any derivative thereof.

In one embodiment, the present invention provides a compound of FormulaI, wherein R¹ or R¹—X represents an aminoglycoside antibiotics moiety,wherein the aminoglycoside antibiotics is selected from the groupconsisting of Gentamicin, Streptomycin, Kanamycin, Fradiomyctn,Paromomycin, Tobramycin, Netilmicin, Amikacin, Neomycin, Ribostamycin,Dibekacin, Sisomicin, Isepamicin, Bekanamycin, Astromicin, Plazomicin,and any derivative thereof.

In one embodiment, the present invention provides a compound of FormulaI, wherein R¹ or R¹—X represents Vancomycin, Linezolid, Erythromycin,Eperezolid, or any derivative thereof.

In one embodiment, the present invention provides a compound of FormulaI, wherein R¹ or R¹—X represents an antibiotics moiety of anantibiotics, and wherein the antibiotics is selected from the groupconsisting of Gentamicin, Streptomycin, Kanamycin, Fradiomyctn,Paromomycin, Tobramycin, Netilmicin, Amikacin, Neomycin, Ribostamycin,Dibekacin, Sisomicin, Isepamicin, Bekanamycin, Astromicin, Plazomicin,Vancomycin, Linezolid, Erythromycin, Eperezolid, and any derivativethereof.

In one embodiment, the present invention provides a compound of FormulaI, wherein R² and R³ are each independently C₁-C₄ branched or unbranchedalkyl chain.

In one embodiment, the present invention provides a compound of FormulaI, wherein R² and R³ are isobutyl group.

In one embodiment, the present invention provides a compound of FormulaI, wherein R⁴ is hydrogen.

In one embodiment, the present invention provides a compound of FormulaI, wherein L₁, L₂, L₃ are each independently C₁-C₄ branched orunbranched alkyl chain.

In one embodiment, the present invention provides a compound of FormulaI, wherein L₁ is —(CH₂)₃—, L₂ is —(CH₂)₃—, and L₃ is —(CH₂)—.

In one embodiment, the present invention provides a compound of FormulaI, wherein R¹ or R¹—X represents an antibiotics moiety of anantibiotics, and wherein the antibiotics is selected from the groupconsisting of Gentamicin, Streptomycin, Kanamycin, Fradiomyctn,Paromomycin, Tobramycin, Netilmicin, Amikacin, Neomycin, Ribostamycin,Dibekacin, Sisomicin, Isepamicin, Bekanamycin, Astromicin, Plazomicin,Vancomycin, Linezolid, Erythromycin, Eperezolid, and any derivativethereof; R² and R³ are isobutyl group; R⁴ is hydrogen; L₁ is —(CH₂)₃—,L₂ is —(CH₂)₃—, and L₃ is —(CH₂)—; X is O; and n is 4.

In one embodiment, the present invention provides a compound of FormulaI, wherein R¹ or R¹—X represents an aminoglycoside antibiotics moiety ofan antibiotics, and wherein the antibiotics is selected from the groupconsisting of Gentamicin, Kanamycin, Tobramycin, Amikacin, Neomycin,Plazomicin, and any derivative thereof; R² and R³ are isobutyl group; R⁴is hydrogen; L₁ is —(CH₂)₃—, L₂ is —(CH₂)₃—, and L₃ is —(CH₂)—; X is O;and n is 4.

In one embodiment, the present invention provides a compound of FormulaI, wherein R¹ or R¹—X represents an aminoglycoside antibiotics moiety,and the aminoglycoside antibiotics is Kanamycin or any derivativethereof; R² and R³ are isobutyl group; R⁴ is hydrogen; L₁ is —(CH₂)₃—,L₂ is —(CH₂)₃—, and L₃ is —(CH₂)—; X is O; and n is 4.

In one embodiment, the present invention provides a compound of FormulaI with the specific structure Formula A, wherein the antibiotic moietyis a Kanamycin moiety:

In another aspect, the present disclosure provides a compound of FormulaII:

or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt,derivative or prodrug thereof, wherein

-   -   R¹ is an antibiotic moiety;    -   R² and R³ are each independently H, a C₁-C₈ branched or        unbranched alkyl chain, or a C₃-C₈ cyclic alkyl;    -   R⁴ is H, a C₁-C₈ branched or unbranched alkyl chain, or a C₃-C₈        cyclic alkyl;    -   L₃ is C₁-C₈ branched or unbranched alkyl chain, or a C₃-C₈        cyclic alkyl;    -   Z is a linker comprising a disulfide bond (S—S bond); and    -   n is 3-8.

In one embodiment, the present invention provides a compound of FormulaII, wherein R¹ represents the moiety of an aminoglycoside antibiotics orany derivative thereof.

In one embodiment, the present invention provides a compound of FormulaII, wherein R¹ represents an aminoglycoside antibiotics moiety, whereinthe aminoglycoside antibiotics is selected from the group consisting ofGentamicin, Streptomycin, Kanamycin, Fradiomyctn, Paromomycin,Tobramycin, Netilmicin, Amikacin, Neomycin, Ribostamycin, Dibekacin,Sisomicin, Isepamicin, Bekanamycin, Astromicin, Plazomicin, and anyderivative thereof.

In one embodiment, the present invention provides a compound of FormulaII, wherein R¹ represents an antibiotics moiety of an antibiotics,wherein the antibiotics is selected from the group consisting ofVancomycin, Linezolid, Erythromycin, Eperezolid, and any derivativethereof.

In one embodiment, the present invention provides a compound of FormulaII, wherein R¹ represents an antibiotics moiety of an antibiotics,wherein the antibiotics is selected from the group consisting ofGentamicin, Streptomycin, Kanamycin, Fradiomyctn, Paromomycin,Tobramycin, Netilmicin, Amikacin, Neomycin, Ribostamycin, Dibekacin,Sisomicin, Isepamicin, Bekanamycin, Astromicin, Plazomicin, Vancomycin,Linezolid, Erythromycin, Eperezolid, and any derivative thereof

In one embodiment, the present invention provides a compound of FormulaII, wherein R² and R³ are each independently C₁-C₄ branched orunbranched alkyl chain.

In one embodiment, the present invention provides a compound of FormulaII, wherein R² and R³ are isobutyl group.

In one embodiment, the present invention provides a compound of FormulaII, wherein R⁴ is hydrogen.

In one embodiment, the present invention provides a compound of FormulaII, wherein L₃ is C₁-C₄ branched or unbranched alkyl chain.

In one embodiment, the present invention provides a compound of FormulaII, wherein L₃ is —(CH₂)—.

In one embodiment, the present invention provides a compound of FormulaII, wherein the linker Z is —(C═O)—(CH₂)₃—S—S—(CH₂)₃—, and the carbonylgroup (C═O) of the linker Z is connected to the antibiotic moiety R¹through an oxygen or nitrogen to form a ester or amide group.

In one embodiment, the present invention provides a compound of FormulaII, wherein n is 4.

In one embodiment, the present invention provides a compound of FormulaII, wherein R¹ represents an aminoglycoside antibiotics moiety of anantibiotics, and wherein the antibiotics is selected from the groupconsisting of Gentamicin, Streptomycin, Kanamycin, Fradiomyctn,Paromomycin, Tobramycin, Netilmicin, Amikacin, Neomycin, Ribostamycin,Dibekacin, Sisomicin, Isepamicin, Bekanamycin, Astromicin, Plazomicin,and any derivative thereof; R² and R³ are isobutyl group; R⁴ ishydrogen; L₃ is —(CH₂)—; Z is —(C═O)—(CH₂)₃—S—S—(CH₂)₃—, and thecarbonyl group (C═O) of the linker Z is connected to the antibioticmoiety R¹ through an oxygen or nitrogen to form a ester or amide group;and n is 4.

In one embodiment, the present invention provides a compound of FormulaII, wherein R¹ represents an antibiotics moiety of an antibiotics, andwherein the antibiotics is selected from the group consisting ofVancomycin, Linezolid, Erythromycin, Eperezolid, and any derivativethereof; R² and R³ are isobutyl group; R⁴ is hydrogen; L₃ is —(CH₂)—; Zis —(C═O)—(CH₂)₃—S—S—(CH₂)₃—, and the carbonyl group (C═O) of the linkerZ is connected to the antibiotic moiety R¹ through an oxygen or nitrogento form a ester or amide group; and n is 4.

In one embodiment, the present invention provides a compound of FormulaII, wherein R¹ represents an antibiotics moiety of an antibiotics, andwherein the antibiotics is selected from the group consisting ofGentamicin, Streptomycin, Kanamycin, Fradiomyctn, Paromomycin,Tobramycin, Netilmicin, Amikacin, Neomycin, Ribostamycin, Dibekacin,Sisomicin, Isepamicin, Bekanamycin, Astromicin, Plazomicin, Vancomycin,Linezolid, Erythromycin, Eperezolid, and any derivative thereof; R² andR³ are isobutyl group; R⁴ is hydrogen; L₃ is —(CH₂)—; Z is—(C═O)—(CH₂)₃—S—S—(CH₂)₃—, and the carbonyl group (C═O) of the linker Zis connected to the antibiotic moiety R¹ through an oxygen or nitrogento form a ester or amide group; and n is 4.

In one embodiment, the present invention provides a compound of FormulaII, wherein R¹ represents an antibiotics moiety of an antibiotics,wherein the antibiotics is selected from the group consisting ofGentamicin, Kanamycin, Tobramycin, Amikacin, Neomycin, Plazomicin, andany derivative thereof; R² and R³ are isobutyl group; R⁴ is hydrogen; L₃is —(CH₂)—; Z is —(C═O)—(CH₂)₃—S—S—(CH₂)₃—, and the carbonyl group (C═O)of the linker Z is connected to the antibiotic moiety R¹ through anoxygen or nitrogen to form a ester or amide group; and n is 4.

In one embodiment, the present invention provides a compound of FormulaII, wherein the linker Z is —X—(C═O)-L₁-S—S-L₂-, wherein L₁ and L₂ areeach independently C₁-C₈ branched or unbranched alkyl chain, or a C₃-C₈cyclic alkyl; X is O or NR⁵, wherein R⁵ is H, C₁-C₈ branched orunbranched alkyl chain, or a C₃-C₈ cyclic alkyl, or X combined with R¹together is an antibiotic moiety.

In one embodiment, the present invention provides a compound of FormulaI or II, wherein R¹ or R¹—X represents Tobramycin moiety or anyderivative thereof.

In one embodiment, the present invention provides a compound of FormulaI or II, wherein the compound is:

or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt,derivative or prodrug thereof.

The present disclosure also provides methods for treating a host cellhaving a microbial infection comprising the steps of administering acompound of Formula I or Formula II, or a stereoisomer, tautomer,solvate, pharmaceutically acceptable salt, derivative or prodrugthereof.

The present disclosure further provides methods for treating a patient,either mammal or animal, having a microbial infection with a compound ofFormula I or Formula II, or a stereoisomer, tautomer, solvate,pharmaceutically acceptable salt, derivative or prodrug thereof.

The present disclosure provides methods for inhibiting formation of abiofilm by a compound of Formula I or Formula II, or a stereoisomer,tautomer, solvate, pharmaceutically acceptable salt, derivative orprodrug thereof.

The present disclosure provides methods for inhibiting growth of anestablished biofilm with a compound of Formula I or Formula II, or astereoisomer, tautomer, solvate, pharmaceutically acceptable salt,derivative or prodrug thereof. In one aspect, the compound used for anymethod is:

or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt,derivative or prodrug thereof.

The compounds of the present invention may be prepared by a variety ofprocedures, some of which are illustrated in the schemes below. Somesubstituents may be eliminated in the following schemes for the sake ofclarity and are not intended to limit the teaching of the schemes in anyway.

The method of preparing Examples 1-3 is illustrated in Scheme 1. Aperson with ordinary skill in the art may appreciate that the method toprepare Examples 1-3, may apply to the method of preparing othercompounds disclosed in the present invention without undue experiment.

Prep.1 (R=Boc) Prep.1′ (R=Cbz)Prep.2 (R=Boc; X═S); Prep.2′ (R=Cbz; X═S); Prep 2″ (R=Boc; X═C)Prep.3 (P14LRR on resin)

Example 1 (R═H; X═S); Example 2 (R═H, X═C); Example 3 (R=Cbz, X═S)Scheme 1: The Method of Preparing Compounds of Formula A

Preparation 1 or 1′, which is a Kanamycin analog with amino groupsprotected by an appropriate nitrogen-protecting group such as atert-butyloxycarbonyl (Boc) group or carboxybenzyl (Cbz), can beprepared by methods disclosed in literatures. See Disney, M. D.;Barrett, O. J. Biochemistry (Mosc.) 2007, 46 (40), 11223; and Chen, G.;Pan, P.; Yao, Y.; Chen, Y.; Meng, X.; Li, Z. Tetrahedron 2008, 64 (38),9078.

For example, Preparations 1 can react with an appropriate acid such as4,4′-dithiodibutyric acid in Step A under an appropriate reactionconditions to provide Preparation 2. Preparation 2 may further reactwith Preparation 3 (resin-bound P14LRR) in Step B, followed by thetreatment of trifluoroacetic acid in Step C to provide Example 1.

The resin-bound P14LRR was synthesized using Fmoc-based, solid phasestrategy on the Rink amide ChemMatrix resin, as previously described.See Fillon, Y. A.; Anderson, J. P.; Chmielewski, J., Cell penetratingagents based on a polyproline helix scaffold Journal of the AmericanChemical Society, 2005, 127, 11798-803. Geisler, I.; Chmielewski, J.,Probing length effects and mechanism of cell penetrating agents mountedon a polyproline helix scaffold. Bioorg. Med. Chem. Lett., 2007, 17,2765-8.

Preparations Preparation 1: di-tert-butyl((1S,3R,4S,5R,6R)-4-(((2S,3R,4S,5S,6R)-4-((tert-butoxycarbonyl)amino)-3,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-6-(((2R,3R,4S,5S,6R)-6-(((tert-butoxycarbonyl)amino)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)-5-hydroxycyclohexane-1,3-diyl)dicarbamate(Tetra-N-(tert-butoxycarbonyl) kanamycin A)

Tetra-N-(tert-butoxycarbonyl) kanamycin A (Preparation 1 is preparedwith method disclosed in Disney, M. D.; Barrett, O. J. Biochemistry(Mosc.) 2007, 46 (40), 11223.)

Preparation 1′

Preparation 1′ was prepared with essentially the same method ofPreparation 1 except the protecting group is carboxybenzyl group (Cbz).

Preparation 2:4-((4-(((2R,3R,4S,5R,6R)-2-(((1R,2R,3S,4R,6S)-4,6-bis((tert-butoxycarbonyl)amino)-3-(((2S,3R,4S,5S,6R)-4-((tert-butoxycarbonyl)amino)-3,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-2-hydroxycylohexyl)oxy)-6-(((tert-butoxycarbonyl)amino)methyl)-3,5-dihydroxytetrahydro-2H-pyran-4-yl)oxy)-4-oxobutyl)disulfanyl)butanoicacid

To a solution of 4,4′-dithiodibutyric acid (5.9 mmol) in 5 mL of drydimethylformamide (DMF), at room temperature, was addedO-(7-azabenzotriazole-1-yl)-N,N,N,N′-tetramethyluroniumhexafluorophosphate (HATU) (5.2 mmol) and diisopropylethylamine (DIEA)(27 mmol). After 20 min at RT, Preparation 1 (2.3 mmol) was added. Themixture was stirred overnight. The solvent was removed in vacuo, and theresulting material was dissolved in dimethylsulfoxide (DMSO) andpurified by reversed phase HPLC using a C8 and C18 semi-preparativecolumn (Phenomenix). An eluent consisting of solvent A (acetonitrile and0.1% trifluoroacetic acid (TFA)) and solvent B (water and 0.1% TFA) witha 60 min gradient of 35-95% solvent A, a flow rate of 10 mL/min and ELSdetection were used for Preparation 2. Fractions consisting of thedesired products were collected and lyophilized to obtain Preparation 2(approximately a 10% yield). The structure was elucidated using 2D NMRmethods COSY, TOCSY and HMBC.

Preparation 2′

Preparation 2′ was prepared with essentially the same method ofPreparation 2 except the protecting group is carboxybenzyl group (Cbz).

Preparation 2″

Preparation 2″ was prepared with essentially the same method ofPreparation 2 except that X is C.

Preparation 3: Resin Bound P14LRR

Preparation 3 was synthesized using Fmoc-based, solid phase strategy onthe Rink amide ChemMatrix resin (45 tmol), as previously describedmethods. The procedure was repeated until all amino acids were coupledto the resin. The resin was washed with dimethylformamide (DMF),dichloromethane (DCM), methanol (MeOH), DCM and DMF (2×5 mL). SeeFillon, Y. A.; Anderson, J. P.; Chmielewski, J., Cell penetrating agentsbased on a polyproline helix scaffold. Journal of the American ChemicalSociety, 2005, 127, 11798-803. Geisler, I.; Chmielewski, J., Probinglength effects and mechanism of cell penetrating agents mounted on apolyproline helix scaffold. Bioorg. Med. Chem. Lett., 2007, 17, 2765-8.

Example 1

Preparation 2 (2 equiv., 0.12 mmol) in DMF (5 mL), HATU (2 equiv., 0.12mmol) and diisopropylethylamine (DIEA) (4 equiv., 0.24 mmol) were addedto the resin Preparation 3 and the reaction flask was agitated for 24 h.The resin was washed with DMF, DCM, MeOH, and DCM (2×5 mL). Atrifluoroacetic acid (TFA) cleavage cocktailTFA:triisopropylsilane:water (95:2.5:2.5) (10 mL) was added to the resinand the mixture was agitated for 1 h. The solution was filtered throughglass wool into a 50 mL centrifuge tube. The resin was washed with DCM(4×4 mL), and the filtrate was collected into the same tube. Theresulting solution was concentrated in vacuo to remove the TFA. Theresidue wa sdissolved in cold diethyl ether and placed in the freezer toprecipitate the desired peptide conjugates. The precipitate wascollected by centrifugation and washed with cold diethyl ether. Thecollected materials was purified to homogeneity by reversed phase HPLCusing C₁₈ semi-preparative column (Phenomenix). An eluent consisting ofsolvent A (acetonitrile and 0.1% TFA) and solvent B (water and 0.1% TFA)with a flow rate of 12 mL/min, UV detection at 214 nm and 60 min solventgradient of 15-60% for Example 1. Expected Mass 3302.87, Observed Mass3303.46.

Examples 2-4 were prepared with essentially the same method of Example1.

TABLE 1 Examples 2-4 Example 2 R = H, X = C Expected Mass 3266.96,Observed Mass 3268.59

Example 3 R = Cbz, X = S Expected Mass 3839.02, Observed Mass 3844.22

Example 4 Expected Mass 3814.5, Observed Mass 3810.17

Example 5

Example 5 was synthesized with the method illustrated in Scheme 2.

Preparation 4

To a flame dried flask equipped with stir bar, Penta-N-Boc Tobramycin(CAS No.: 172950-21-9; 101.3 mg, 0.105 mmol),4-(2-pyridyldithio)butanoic acid (20 mg, 0.087 mmol),diisopropylethylamine (DIEA) (182 μL, 1.05 mmol), HATU (24.5 mg, 0.105mmol) were added with 4 Å molecular sieves in 500 μL dry N, N′dimethylformamide (DMF). The reaction was stirred for 72 hours andreverse phase HPLC was used to determine reaction completeness. Thereaction components were purified to homogeneity via reverse phase HPLC(C18, 55-75% CH₃CN in water with 0.1% TFA, 254 nm), to afford 2 majoresterification isomers, “A” and “B”, as white powders with yields of 7.9mg (6.4%) and 17 mg (13.8%), respectively. MS (ESI) m/z: calculated[M+H]: 1179.5, Observed: 1179.6.

Isomer A (5.9 mg, 0.005 mmol), was placed in a vial equipped with stirbar, and 139 μL each of dichloromethane (DCM) and trifluoroacetic acid(TFA) were added. The reaction was monitored by TLC (30% methanol:70%dichloromethane) and was judged complete after 1.5 hour. The reactionmixture was concentrated under reduced pressure. Reaction mixture wasresuspended in methanol (250 μL, 2×) and dichloromethane (250 μL, 2×)and the solvents removed under reduced pressure to yield a white solid.The formation of the desired material was confirmed by massspectrometry, and used in the next step without further purification.Yield: 6.5 mg (79.3%). MS (ESI) m/z: calculated [M+H]: 679.3, Observed:679.3. Structure of Preparation 4 was confirmed by 1D- and 2D-NMRtechniques, including H¹, C¹³, DEPT-135, COSYPR, TOCSYPR, HMBC and HSQC.

Example 5

To an Eppendorf tube containing the P14LRR-SH peptide (1 mg, 0.3 μmol)in degassed water (186 μL, pH 9) was added dropwise Preparation 4 (2.3mg, 3 μmol) in degassed water (117 μL, pH 9). The reaction was allowedto react overnight, and the reaction progress was monitored with reversephase HPLC. The desired conjugate Example 5 was purified to homogeneityvia reverse phase HPLC (C18, 20-80% CH₃CN in water with 0.1% TFA, 214nm). The product fractions were combined, acetonitrile was removed underreduced pressure and the resulting aqueous solution was lyophilized toafford an off white solid Example 5. Yield: 0.2 mg (20%). MS (MALDI)m/z: calculated: 3285.9, observed: 3285.3.

Characterization of the Conjugates—Analytical HPLC

Purity was determined by analytical RP-HPLC using a C18 reverse phaseanalytical column (5 μm, 4 mmx 250 mm; Phenomenex Luna), a flow rate of1.2 mL/min, UV detection at 214 nm, and a 30 mingradient of 15-55%,25-55% and 30-70% solvent A (A: acetonitrile and 0.05% trifluoroaceticacid (TFA) gradient of 15-55%, 25-55% and 30-70% solvent A (A:acetonitrile and 0.05% trifluoroacetic acid (TFA); B: water and 0.05%TFA).

The dual antibiotic conjugate Example 1 was designed to enter mammaliancells and release kanamycin and the peptide within an intracellularreducing environment. To evaluate the half-life of release of theseagents under reducing conditions, Example 3 was constructed as it willrelease the UV-active fragment. The conjugate Example 3 (100 μM in a 1:1mixture of PBS buffer and DMF) was treated with 1 mM dithiothreitol(DTT) and the accumulation of kanamycin fragment was monitored by HPLCand LC-MS. Efficient release from the conjugate with a half-life of1.5±0.2 h and full release after 4 h was observed.

Characterization of the Conjugates—Mass Spectrometry

Peptides were further characterized using matrix associated laserdesorption ionization-time of flight (MALDI-TOF) mass spectrometry(Voyager DE, Applied Biosystems).

Method for Example 1 Stability Studies Using Porcine Liver Esterase

To a solution of PBS, pH 7.4 (690 μL PBS) was added, 10 μL of 10 mMExample 1 (to give a final concentration of 100 μM) and 300 μL ofporcine liver esterase in PBS, pH 7.4 (to give a final concentration of100 unit/ml). The mixture was incubated at 37° C. At different timepoints, 50 μL aliquots from the reaction mixture was removed and 50 μLof cold acetonitrile was added to deactivate the proteins. The solutionwas then vortexed for 20 sec and centrifuged for 10 min at 2000 rpm.Next, 50 μL of the supernatant was removed, added to 50 μL of 100 mMN-α-Benzoyl-L-arginine ethyl ester hydrochloride (BAEE) (Alfa AesarA18181) (to give final concentration 500 μM) as an internal standard,and was immediately injected on an HPLC equipped with a C₁₈ reversephase analytical column (5 μm, 4 mm×250 mm; Phenomenex Luna) with aneluent consisting of solvent A (acetonitrile and 0.1% trifluoroaceticacid (TFA)) and solvent B (water and 0.1% TFA) with a 30 min gradient of15-65% solvent A, a flow rate of 1.2 mL/min and UV detection at 214 nm.The appearance of the peak corresponding to P14LRR modified with4,4′-dithiodibutyric acid was monitored and only ˜15% release wasobserved after 120 hrs. Therefore, Example 1 is not susceptible toesterase cleavage likely due to steric crowding on kanamycin around theester bond.

Method for In Vitro Antimicrobial Activity Assessment

a) Against Escherichia coli and Staphylococcus aureus

Escherichia coli (ATCC 25922) or Staphylococcus aureus (ATCC 25923) wasgrown to the mid-exponential phase in Tryptic Soy Broth (TSB) at 37° C.with shaking. An aliquot of the bacterial suspension was centrifuged for5 min at 3000 rpm, the supernatant aspirated and the pellet re-suspendedin Muller Hinton Broth (MHB) to a final optical density of 0.001 asmeasured by absorbance at 600 nm (OD₆₀₀). Next 90 μL of this culture wasadded in a sterile 96-well plate (Cellstar 655180) and then supplementedwith 10 μL of sterile water or two-fold serial dilutions of the drugs inwater. Melittin was used as positive control for both bacterial strains.The plate was then incubated for 6 h at 37° C. The OD₅₉₀ was determinedusing a microplate reader (TECAN SpectraFluor Plus). The minimuminhibitory concentration (MIC) was defined as the lowest concentrationof drug at which no growth was observed. Data was obtained in duplicatesfrom at least two independent experiments.

Against Pathogenic Bacteria

The broth microdilution technique was used to determine the minimuminhibitory concentrations (MIC) of drugs according to the guidelines ofthe Clinical and Laboratory Standards Institute (CLSI). Salmonellaenteritidis, Shigella flexneri, Brucella abortus, or Mycobacteriumsmegmatis were diluted to a bacterial inoculum of 5×10⁵ colony formingunit (CFU/mL) in Mueller-Hinton broth (MHB) (S. enteritidis, and S.flexneri), Brain Heart Infusion broth (BHI) (B. abortus), and TrypticSoy Broth (TSB) (M. smegmatis). Drugs were added to polystyrene 96 wellplates (CytoOne, CC7672-7596) at desired concentrations and plates wereincubated at 37° C. for 18 h (S. flexneri), 24 h (S. enteritidis) or 48h (B. abortus and M. smegmatis). MIC was defined as the lowestconcentration of drug which inhibited the visible growth of bacteria.

The resazurin microtiter assay (REMA) was used to determine the minimuminhibitory concentrations (MIC) of drugs against Mycobacteriumtuberculosis H37Ra. Briefly, 100 μL of Middlebrook 7H9 brothsupplemented with Oleic Albumin Dextrose Catalase Growth Supplement(OADC) enrichment and glycerol was added to wells of 96-well plate, andserial two fold dilutions of each drug were prepared directly in theplate. One hundred microliters of inoculum was added to each well. Agrowth control and a sterile control were also included for eachisolate. The plate was covered, sealed in a plastic bag, and incubatedat 37° C. under a normal atmosphere. After 7 days of incubation, 30 μLof resazurin solution was added to each well, and the plate wasreincubated for 24 hr. A change in color from blue to pink indicatedbacterial growth, and the MIC was determined as the minimumconcentration of drug that prevented color change.

c) Determination of Minimum Inhibitory Concentration (MIC) ValuesAgainst Biofilmforming Clinical Isolates of S. aureus and S. epidermidis

Biofilm-forming clinical isolates of S. aureus (ATCC 6538) and S.epidermidis (ATCC 35984) were grown overnight in TSB. After incubation,cultures were diluted to 5×10⁵ colony forming unit (CFU/ml) in MHB. Theminimum inhibitory concentrations of compounds were determined via thebroth microdilution technique according to the guidelines of theClinical and Laboratory Standards Institute (CLSI). Compounds were addedto polystyrene 96-well plates at desired concentrations and subsequentlyserially diluted. The MIC was defined as the lowest concentration ofpeptide or antibiotic that inhibited bacterial growth visually.

TABLE 2 MIC values [μM] for the antibacterial activity of the dualconjugates compared to individual component. E. S. S. B. S. M. M. S. S.coli aureus enteritidis abortus flexneri smegmatis tuberculosis aureus^([c]) epidermis ^([c]) P14LRR 4 16 32 16 8 8 16 16 8 P14SH 4 16 4 2 4 48 — — kanamycin 2 2 16 4 2 8 2 4 >128 P14LRR:kanamycin^([b]) 2 2 8 4 2 41 — — Example 1 2 2 2 0.12 1 1 1 1 1 Example 2 2 16 8 2 2 2 1 — —^([a])The minimum inhibitory concentration, ^([b])1:1 mixture of P14LRRand kanamycin ^([c])clinical isolate that forms biofilms

Antimicrobial Activity

The antibacterial activity of kanamycin and the conjugate wasinvestigated against ESKAPE pathogens. The peptide, P14LRR, alonedisplayed moderate to weak antibacterial activity against ESKAPEpathogens with MIC values ranging from 8 to 64 μM (Table 1). Kanamycinwas only active against two strains (S. aureus ATCC 6538 and MRSAUSA400). However, other bacteria were resistant to kanamycin (MIC, 128to >256 μM). Example 1 demonstrated a very potent, broad-spectrumantibacterial activity over kanamycin and the peptide (P14LRR). The MICof Example 1 was 1 to 2 μM against all strains tested. This represents a2-4 fold improvement in the MIC relative to kanamycin againstkanamycin-sensitive strains and 128 to >256-fold improvement in the MICrelative to kanamycin against kanamycin-resistant strains.Interestingly, Example 1 demonstrated potent activity against severalmultidrug-resistant clinical isolates including methicillin-resistant S.aureus (MRSA USA400), methicillin-resistant S. epidermidis (MRSE) andvancomycin-resistant E. faecium (VRE) (Table 1). Of particular note,Example 1 demonstrated potent activity against A. baumannii ATCCBAA-1605, a multidrug-resistant isolate obtained from the sputum of aCanadian soldier serving in Afghanistan. This strain is resistant tonumerous antibiotics including kanamycin, meropenem, imipenem,ceftazidime, ciprofloxacin, piperacillin, ticarcillin, cefepime,aztreonam and gentamicin (Table 1). Moreover, Example 1 demonstratedpotent activity against colistin-resistant P. aeruginosa (1109) isolatedfrom a cystic fibrosis patient. This isolate exhibits high-levelresistance to colistin (MIC=128 μM), kanamycin and several antimicrobialpeptides. Colistin is an antibiotic of last resort formultidrug-resistant P. aeruginosa infections. The emergence of resistantisolates to colistin, particularly via plasmid-mediated resistance inEnterobacteriaceae, highlights the urgent need to discover newantimicrobial agents to address this issue. The discovery that Example 1has potent activity against a colistin-resistant P. aeruginosa strainindicates a potential therapeutic advantage of P14KanS over severalantimicrobials that are ineffective against colistin-resistant P.aeruginosa.

TABLE 3 Minimum inhibitory concentration (MIC) (μM) of compounds againstclinical and drug-resistant bacterial isolates Vancomycin- Methicillin-Colistin- resistant resistant S. aureus S. epidermidis P. aeruginosaresistant A. baumannii K. pneumoniae E. faecium S. aureus ATCC ATCC ATCCP. aeruginosa BAA- BAA- ATCC Compound USA400 6538 35984 PAO1 1109 16051706 700221 Phenotype Resistant to Biofilm Prototype Biofilm Isolated AResistant to Resistant to methicillin forming biofilm producing from acystic multidrug- kanamycin vancomycin, and strain, producer, strain.fibrosis resistant teicoplanin tetracycline methicillin resistant toResistant to patient. strain; and sensitive methicillin, kanamycinResistant to resistant to kanamycin gentamicin, colistin and kanamycin,kanamycin, kanamycin ceftazidime, erythromycin, gentamicin, clindamycinticarcillin, and piperacillin, trimethoprim aztreonam, cefepime,ciprofloxacin, imipenem, and meropemem P14LRR 64 16 32 64 64 32 32 8Kanamycin 4 2 >256 >256 >256 128 256 >256 Example 1 1 1 1 2 2 1 2 2 Fold4 2 >256 >128 >128 128 128 >128 enhancement in MIC compared to kanamycin

Antimicrobial activities of the Example 1 and Example 2 conjugates wereevaluated with a series of Gram positive and negative bacteria,including a range of intracellularpathogens (Table 1). This activity wascompared to that obtained for P14LRR, P14SH, kanamycin and a 1:1 mixtureof kanamycin and P14LRR. Across the series of bacteria P14KanS is highlyactive with MIC values ranging from 0.12 to 2 μM, including M.tuberculosis. With four strains of bacteria P14KanS is 2- to 32-timesmore active, respectively, than the non-covalent mixture of P14LRR andkanamycin. With the exception of E. coli and M. tuberculosis, thisconjugate is also 2- to 16-fold more potent than the non-reducibleP14KanC conjugate. The activity of P14KanS was also evaluated onclinical isolates of S. aureus and S. epidermis that form biofilms, andthe conjugate was 8- to 16-fold more potent than the P14LRR peptide,respectively. It was aslo investigated if the Example 1 and Example 2conjugates disrupted bacteria membranes by monitoring beta-galactosidaserelease from E. coli upon addition of the conjugates. At five timestheir MIC values, no significant release of beta-galactosidase wasobserved, whereas melittin at 5 times its MIC displayed a substantiallevel of release as has previously been shown. These data demonstratethat the kanamycin-peptide conjugates do not lyse membranes as themechanism for their antibacterial activity, as was observed for P14LRR.

These data illustrate that neither kanamycin nor P14LRR is capable ofeffectively clearing intracellular bacteria on their own, even whenco-administered. Only Example 1 conjugate potently clears intracellularpathogens by taking advantage of the non-membrane lytic, mammalian cellpenetrating activity of P14LRR to co-deliver kanamycin. When comparingthe antimicrobial activity of Example 1 against Salmonella and Brucella,Example 1 is 16-fold more potent against Brucella in vitro (Table 1),but the activity trends switches in cyto with a more effective clearanceof Salmonella intracellularly. Both of these pathogens enter macrophagesvia vacuoles, but ultimately reside in sub-cellular locations such asendosomes and the endoplasmic reticulum, respectively. Since Example 1localizes to endosomes, it is reasonable that this agent is moreeffective against Salmonella within mammalian cells. The notabledifference in potency between Example 1 and Example 1 demonstrates thatit is crucial for the two antibiotics to separate and go their own waywithin macrophages for optimal performance.

Method for Cytotoxicity Assessment

Cellular toxicity was assessed using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cellviability assay. Briefly, 1.5×10⁴ J774A.1 cells suspended in 200 μL, ofDulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heatinactivated Fetal Bovine Serum (FBS), 1% Lglutamine and 1%penicillin/streptomycin were seeded in 96-well plates and incubated at37° C. in a 5% CO2 atmosphere. The cells were cultured for 24 h (60%confluency) before the assays. At the end of the pre-incubation period,the media was discarded and the cells were washed twice with 100 μL PBS.The J774A.1 cells were further incubated with 100 μL of dilutionsranging from 2.5-30 μM of drugs in DMEM for 9 h. Following incubation,20 μL of 5 mg/mL MTT (Sigma M5655) in PBS was added per well and theplate was returned to the incubator for an additional 1.5 h. Next, theMTT solution was aspirated and 100 μL of dimethyl sulfoxide (DMSO) perwell was added to dissolve formazan crystals formed. For eachexperiment, a negative control of untreated cells was also analyzed. Allsamples were run in duplicate, and each experiment was duplicated. Theintensity of color was quantified at 590 nm using a 96-well ELISA platereader (SoftMax ProInc., USA). Results were expressed as the percentagemean absorbance of treated cells in respect to incubation with control.The conjugates and the components display limited cytotoxicity toJ774A.1 cells across a range of concentrations.

Method for Rescue of J774A.1 Cells Infected with Pathogenic Bacteria

Macrophage-like cell line (J774A.1) was seeded at a density of 1.5×10⁴per well in a tissue culture 96-well plate (CytoOne, CC7682-7596) inDulbecco's Modified Eagle Medium (DMEM) media supplemented with 10%Fetal Bovine Serum (FBS), and incubated at 37° C. in a 5% CO₂ atmospherefor 24 h (to 95% confluence). Following incubation, the cells werewashed once with DMEM media. Then the cells were infected withSalmonella enteritidis, Shigella flexneri or Brucella abortus (atmultiplicity of infection 1:10 for S. enteritidis, and 1:100 for S.flexneri or B. abortus) in DMEM and 10% FBS for 1 h. While forMycobacterium tuberculosis cells were infected (at multiplicity ofinfection 1:20) in DMEM and 10% FBS for 4 hr, and for Mycobacteriumsmegmatis cells were infected (at multiplicity of infection 1:50) inDMEM and 10% FBS for 1 h. After infection the wells were washed threetimes with 200 μL media with gentamicin (final concentration 50 μg/mL)and further incubated for 30 minutes (or 1 h for M. tuberculosis) withgentamicin to kill extracellular bacteria. Drugs were diluted in DMEMand 10 FBS % to the desired concentrations. Subsequently, after washing,wells were treated with 100 μL of DMEM and 10% FBS containing drugs for9 h (S. enteritidis and S. flexneri), 12 h (M. smegmatis), 21 h (B.abortus) and 7 days (M. tuberculosis). Ciprofloxacin (5 μM) was used asa positive control for S. enteritidis and B. abortus, and isoniazid(0.12 μM) for M. tuberculosis and M. smegmatis. Medium alone used as anegative control. After incubation, the media was aspirated and thewells were washed twice with PBS. Then 100 μL of PBS with 0.01% triton Xwas added to each well to lyse the macrophage cells. Subsequently,bacteria were diluted 10 fold serially in PBS and plated on Tryptic SoyAgar (TSA) plates (M. tuberculosis was plated on Middlebrook 7H10 Agarplates supplemented with OADC Enrichment). Plates were incubated at 37°C. for 20 h (S. enteritidis and S. flexneri), 48 h (B. abortus), 72 h(M. smegmatis) and 4-5 weeks (M. tuberculosis). After incubation,bacteria were counted and analyzed by Graph pad software. Each drugtreatment was done with three biological replicates. Experiments wererepeated independently twice except M. tuberculosis was done once withthree biological replicates.

Antimicrobial Efficacy of Treatment In Vivo Using a Salmonella-InfectedC. Elegans Animal Model.

The infection and treatment of Caenorhabditis elegans were performed asreported previously. See Alajlouni, R A, Seleem M N. Targeting listeriamonocytogenes rpoA and rpoD genes using peptide nucleic acids. Nucleicacid therapeutics, 2013, 23, 363-7. A pathogen sensitive strain of C.elegans {glp-4(bn2) I; sek-1(km4)} was used in this study. Bacteriallawns used for the C. elegans infection assays were prepared byspreading 200 μL of the overnight culture of Salmonella enteritidis onthe modified Nematode Growth Media (NGM) (0.35% peptone) agar plates.The plates were incubated at 37° C. overnight before being seeded withyoung adult hermaphrodite nematodes, grown at 25° C., from asynchronized culture. The infections were performed at 25° C. for 24 h.Subsequently, the worms were collected and washed with PBS to removeextracellular bacteria and diluted to reach 1 worm per 1 μL of PBS.Approximately 100 worms (100 μL PBS) were transferred to 1.5 mLmicrocentrifuge tubes. Drugs at concentration equal to 50 μM (exceptciprofloxacin 10 μM) were added to tubes in triplicates. Controlnegative tubes contain only PBS. After 24 h, worms were checked forsurvival under a microscope to assess the toxicity of drugs. The tubeswere centrifuged and the supernatant was removed. Worms were washedtwice with 1 ml of PBS then 200 μg of autoclaved silica carbide wereadded to each tube, and the worms were vortexed for 1 minute at highspeed. One hundred microliter from each tube was diluted 10 foldserially in PBS. Bacteria were plated in Tryptic Soy Agar (TSA) platescontaining spectinomycin (18 μg/mL) to allow for selective growth of S.enteritidis over Escherichia coli. Plates were incubated for 16 h at 37°C. and colonies were counted. Statistical analysis was analyzed by twotailed student t test. (P≤0.05 was considered significant).

Method for In Vivo Toxicity Assessment in C. elegans Animal Model

Bacterial lawns used for C. elegans infection assays were prepared byspreading 200 ul of the overnight culture of biofilm strains of S.aureus or S. epidermidis on modified NGM (0.35% peptone) agar plates.The plates were incubated at 37° C. overnight before being seeded withyoung adult hermaphrodite nematodes, grown at 25° C., from asynchronized culture. The infections were performed at 25° C. for 12hours. After infection of synchronized worms, around 25-30 adult wormswere transferred to each well in 96 well plate, Drugs at concentrationequal to 50 μM were added in triplicates. Control negative contains onlyPBS. Worms were checked for survival under microscope at different timepoints. Living nematodes maintain a sinusoidal shape, whereas deadnematodes appear as straight, rigid rods as the corpse becomes filledwith bacteria.

Encouraged by the potent antibacterial activity of the peptide conjugateExample 1 in vitro and in the in cyto bacterial clearing assay, it wasinvestigated the ability of the conjugate to clear S. enteritidis usingan in vivo C. elegans model. First the toxicity of Example 1, P14LRR,kanamycin and melittin at 50 μM against C. elegans were evaluated. After3 days of treatment with Example 1, P14LRR or kanamycin the C. eleganswere highly viable (>90%), whereas no live worms were observed withmelittin after this time. These data suggested the non-toxic nature ofthe kanamycin-peptide conjugate. To monitor antibacterial activity, S.enteritidis infected C. elegans were treated with a range ofconcentrations of Example 1, P14LRR, kanamycin, and a 1:1 mixture ofP14LRR and kanamycin for 24 hr, and the bacteria levels were monitored.Example 1 demonstrated significantly reduced levels of Salmonella intheC. elegans host as compared to the 1:1 mixture of antibiotics across allconcentrations, and at the highest dose approximately 90% of thebacteria had been cleared in vivo. The C. elegans infection modelconfirms that Example 1 does exhibit potent in vivo antimicrobialactivity and has significant promise to be used as a novel treatment ofintracellular pathogens.

Method for Leakage of β-Galactosidase from E. coli Treated with Example1

In a sterile 250 mL erlenmeyer flask E. coli (ATCC 25922) was grown tomid-exponential phase (OD590≈0.6) in TSB (˜50 mL) at 37° C. withshaking. β-Galactosidase expression was induced by the addition offreshly prepared isopropyl-β-D-thiogalactopyranoside (IPTG) (GoldBiotechnology I2481C5) in PBS (1 mM final concentration). A 4 mL aliquotof the bacterial suspension was centrifuged, washed twice with fresh TSBand plated into a sterile 96-well plate (90 μL). Next, 10 μL aliquots ofExample 1 in sterile water were added to give final concentrations of 10μM. Bacteria treated with sterile water and melittin (40 μM finalconcentration) served as controls. The plate was then incubated for 1 hat 37° C. At the end of the incubation period, the plate was centrifugedat 3000 rpm at 4° C. for 10 min. 80 μL of the supernatant from each wellwas carefully transferred to a new sterile 96-well plate. Next, 20 μL offreshly prepared 2-Nitrophenyl-β-D-galactopyranoside (ONPG) (Sigma73660) in PBS was added to each well (0.8 mg/ml final concentration).The β-Galactosidase activity was monitored at OD₄₀₅ every five minutesfor a period of 1 h using a micro-plate reader. Data was obtained induplicates from at least two independent experiments.

Anti-Inflammatory Effect of Compounds on LPS Stimulated Macrophages(Neutralizing the Effect of LPS on Macrophages).

The anti-inflammatory effect of compounds on LPS stimulated macrophages,J774A.1 cells was investigated. Next, the cells were stimulated with LPS(150 ng/ml final concentration) in the presence of 10 μM of compounds.Cells that were stimulated with LPS alone and untreated cells served ascontrols. Cells were incubated for six hours at 37° C. and supernatantsfrom each treatment were collected and stored at −20° C. until use.Cytokine detection of tumor necrosis factor-α (TNF-α) and interleukin-6(IL-6) in supernatants was done using ELISA as described beforefollowing the manufacturer's instructions^(24,25). Cytokine levels wereexpressed as percent change relative to the LPS-stimulated control,using triplicate samples for each treatment condition.

Example 1 and the peptide P14LRR were able to bind to LPS in vitro asevident by inhibition of the LPS-induced activation of the LAL enzyme(Limulus amoebocyte lysate). Example 1, P14LRR and colistin (a knownLPS-binding agent) produced 60.8%±6.5, 71.2%±5 and 79.76%±4.6 inhibitionof the LAL enzyme at 10 μM, respectively. However, kanamycindemonstrated only minimal inhibition (6.9%±3.5) at the sameconcentration.

Example 1 and P14LRR were able to inhibit LPS-induced proinflammatorycytokines in macrophages in a manner similar to colistin. Example 1,P14LRR and colistin, at 10 μM, decreased TNF-α levels by 65.53%±2.45,68.4%±5.43, 97.53%±0.32, respectively; and IL-6 levels by 74.71%±9.10,81.92%±0.78 and 95.04%±0.56%, respectively. Kanamycin, in contrast,produced an 11.41%±4.5 and 10%±3.30 inhibition of TNF-α and IL-6 levels,respectively. The capability of Example 1 to reduce endotoxin-mediatedproinflammatory cytokine production provides a potential avenue for itsdevelopment as an antibacterial agent to treat sepsis and also as anadjunctive with antibiotics to overcome sepsis.

Efficacy of Compounds on Bacterial Biofilms

The compounds of the present disclosure were examined for their abilityto disrupt bacterial biofilms using the microtiter dish biofilmformation assay. Briefly, overnight cultures of S. aureus (ATCC 6538)and S. epidermidis (ATCC 35984) were diluted 1:100 in TSB supplementedwith 1% glucose. Overnight cultures of P. aeruginosa PAO1 and A.baumannii ATCC BAA-1605 were diluted 1:100 in M63 minimal mediumsupplemented with magnesium sulfate, glucose and casamino acids.Bacterial suspensions were incubated in 96-well plates at 37° C. for 24hours. After removing media, wells were rinsed with PBS to removeplanktonic bacteria before re-filling wells with fresh media. Compoundswere added at specific concentrations and plates were incubated at 37°C. for 24 hours. After incubation, wells were washed and biofilms werestained with 0.5% (w/v) crystal violet for 30 minutes. The dye wassolubilized with ethanol (95%) and the biofilm mass was quantified atOD₅₉₅. Experiments were done in triplicate and repeated twiceindependently.

Efficacy of P14KanS on Established Biofilms

Biofilms are aggregated bacterial communities covered by apolysaccharide matrix that protects bacteria from host immune defensesand hinders antibiotics from targeting deep-seated bacteria encasedwithin the biofilm. Furthermore, biofilms act as an infectious nichewith sustained release of bacteria inside the host that leads to diseaserelapses and therapy failure. Biofilm development has been linked toserious infections including pneumonia in cystic fibrosis patients,colonization of medical devices, and urinary tract infections. Biofilmspose a serious medical challenge that is difficult to control and thereis a critical need to find agents that can address this problem. Thecapability of Example 1 and control antibiotics to disrupt establishedbiofilms of four different bacterial isolates responsible for majorbiofilm infections—S. aureus, S. epidermidis, P. aeruginosa and A.baumannii were investigated. Example 1 disrupted mature (24 hour)biofilms more potently than several antibiotics of choice. Example 1 wassuperior to kanamycin and the peptide P14LRR against the kanamycinsensitive isolate (S. aureus). Example 1 demonstrated aconcentration-dependent biofilm-disruptive activity. Example 1, at a lowconcentration (1 μM), disrupted more than 65% of the biofilm mass of S.aureus (p<0.05) whereas kanamycin and P14LRR were not effective at thesame concentrations. At higher concentrations, Example 1 disrupted morethan 75% and 82% of biofilm mass of S. aureus, at 4 and 8 μM,respectively, (p<0.05). Kanamycin, vancomycin and linezolid, at 8 μM,reduced approximately 50% of biofilm mass (p<0.05); whereas, P14LRR wasnot effective.

It was also investigated for the efficacy of Example 1 against biofilmsof a clinical multidrug-resistant strain of S. epidermidis ATCC 35984, ahigh slime producing strain that was isolated from septicemic patientswith colonized intravascular catheters in Tennessee, US. This strain isresistant to several antibiotics including methicillin, erythromycin,kanamycin, gentamicin, clindamycin and trimethoprim. The great thicknessof the exopolysaccharide matrix of S. epidermidis biofilms cause it tobecome extremely resilient to penetration by antibiotics. Indeed, maturebiofilms of S. epidermidis were less susceptible to vancomycin andlinezolid even at 64 μM (equivalent to 64-128×MIC) where only 25-30%biofilm mass reduction was observed (FIG. 5b). However, Example 1disrupted more than 30%, 50%, 70%, and 80% of biofilm mass at 8, 16, 32and 64 μM, respectively, (P<0.05). The peptide alone, P14LRR, wasineffective except at 64 μM, disrupting approximately 50% of the biofilmmass. Kanamycin did not disrupt or reduce the biofilm mass of S.epidermidis.

Remarkably, Example 1 was capable of eradicating P. aeruginosa and A.baumannii biofilms at 32 μM, however, kanamycin was not effective.P14LRR (32 μM) was ineffective against P. aeruginosa biofilms anddemonstrated only 30% reduction of A. baumannii biofilm mass.Collectively, the data demonstrate that Example 1 is superior toconventional antibiotics in penetrating and disrupting adherent biofilmsof both Gram-positive and Gram-negative pathogens.

Example 1 exhibited potent antimicrobial activity against ESKAPEpathogens. Example 1 demonstrated a ≥128-fold improvement in MICrelative to kanamycin against kanamycin-resistant strains. Mechanisticstudies confirmed that Example 1 exerts its antibacterial effect byselectively disrupting the bacterial cell membrane. Example 1 was nottoxic or hemolytic at concentrations much higher than its MIC. Unlikemany antibiotics, Example 1 demonstrated rapid bactericidal activityagainst stationary phases of both Gram-positive and Gram-negativepathogens. Furthermore, Example 1 was superior in disrupting adherentbacterial biofilms and in killing intracellular pathogens as compared toconventional antibiotics. It should be noted that Example 1 demonstratedpotent anti-inflammatory activity via the suppression of LPS-inducedproinflammatory cytokines. Finally, Example 1 protected C. elegans fromlethal infections of both Gram-positive and Gram-negative pathogens.

Those skilled in the art will recognize that numerous modifications canbe made to the specific implementations described above. Theimplementations should not be limited to the particular limitationsdescribed. Other implementations may be possible.

We claim:
 1. A method of treating a microbial infection in a patient,wherein the method comprises administering to the patient apharmaceutical composition comprising a compound of formula:

a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, orderivative thereof, wherein R¹ is an antibiotic moiety; R² and R³ areeach independently H, a C₁-C₈ branched or unbranched alkyl chain, or aC₃-C₈ cyclic alkyl; R⁴ is H, a C₁-C₈ branched or unbranched alkyl chain,or a C₃-C₈ cyclic alkyl; L₃ is C₁-C₈ branched or unbranched alkyl chain,or a C₃-C₈ cyclic alkyl; Z is a linker comprising a disulfide bond (S—Sbond); and n is 3-8, wherein the microbial infection is an infectioncaused by pathogen S. aureus, S. epidermidis, P. aeruginosa, A.baumannii, K. pneumoniae, E. faecium, enterobacteriaceae, or anycombination thereof.
 2. The method of claim 1, wherein the compound is:

or a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt,or a derivative thereof, wherein R¹ is an antibiotic moiety; R² and R³are each independently H, a C₁-C₈ branched or unbranched alkyl chain, ora C₃-C₈ cyclic alkyl; R⁴ is H, a C₁-C₈ branched or unbranched alkylchain, or a C₃-C₈ cyclic alkyl; L₁, L₂, L₃ are each independently C₁-C₈branched or unbranched alkyl chain, or a C₃-C₈ cyclic alkyl; X is O orNR⁵, wherein R⁵ is H, C₁-C₈ branched or unbranched alkyl chain, or aC₃-C₈ cyclic alkyl, or X combined with R¹ together is an antibioticmoiety; and n is 3-8.
 3. The method of claim 1, wherein R¹ or R¹—Xrepresents the moiety of an aminoglycoside antibiotics or any derivativethereof.
 4. The method of claim 1, wherein R¹ or R¹—X represents anantibiotic moiety, wherein the antibiotic moiety is of an antibioticsselected from the group consisting of Gentamicin, Streptomycin,Kanamycin, Fradiomyctn, Paromomycin, Tobramycin, Netilmicin, Amikacin,Neomycin, Ribostamycin, Dibekacin, Sisomicin, Isepamicin, Bekanamycin,Astromicin, Plazomicin, Vancomycin, Linezolid, Erythromycin, Eperezolid,and any derivative thereof.
 5. The method of claim 1, wherein R² and R³are each independently C₁-C₄ branched or unbranched alkyl chain.
 6. Themethod of claim 1, wherein R² and R³ are isobutyl group.
 7. The methodof claim 1, wherein R⁴ is hydrogen.
 8. The method of claim 2, wherein L₁is —(CH₂)₃—, L₂ is —(CH₂)₃—, and L₃ is —(CH₂)—.
 9. The method of claim1, wherein R¹ represents an antibiotic moiety, and the antibiotic moietyis of an antibiotics selected from the group consisting of Gentamicin,Streptomycin, Kanamycin, Fradiomyctn, Paromomycin, Tobramycin,Netilmicin, Amikacin, Neomycin, Ribostamycin, Dibekacin, Sisomicin,Isepamicin, Bekanamycin, Astromicin, Plazomicin, Vancomycin, Linezolid,Erythromycin, Eperezolid, and any derivative thereof; R² and R³ areisobutyl group; R⁴ is hydrogen; L₁ is —(CH₂)₃—, L₂ is —(CH₂)₃—, and L₃is —(CH₂)—; X is O; and n is
 4. 10. The method of claim 2, wherein R¹ orR¹—X represents an aminoglycoside antibiotics moiety, and theaminoglycoside antibiotics is selected from the group consisting ofGentamicin, Kanamycin, Tobramycin, Amikacin, Neomycin, Plazomicin, andany derivative thereof; R² and R³ are isobutyl group; R⁴ is hydrogen; L₁is —(CH₂)₃—, L₂ is —(CH₂)₃—, and L₃ is —(CH₂)—; X is O; and n is
 4. 11.The method of claim 2, wherein R¹ or R¹—X represents Kanamycin moiety orany derivative thereof; R² and R³ are isobutyl group; R⁴ is hydrogen; L₁is —(CH₂)₃—, L₂ is —(CH₂)₃—, and L₃ is —(CH₂)—; X is O; and n is
 4. 12.The method of claim 1, wherein the compound is:

a stereoisomer, tautomer, solvate, pharmaceutically acceptable salt, ora derivative thereof.
 13. The method of claim 1, wherein the microbialinfection is a biofilm infection.
 14. The method of claim 1, wherein themicrobial infection is an infection caused by pathogen S. aureus (ATCC6538), S. aureus (USA400), S. epidermidis (ATCC 35984), P. aeruginosa(PAO1), P. aeruginosa (1109), A. baumannii (BAA-1605), K. pneumoniae(BAA-1706), E. faecium (ATCC 700221) or any combination thereof.